Bile acids and derivatives thereof as anorectic agents

ABSTRACT

CERTAIN UNCONJUGATED BILE ACIDS AS WELL AS CERTAIN CONJUGATES THEREOF, AT SUFFICIENT DOSE LEVELS, SUPPRESS THE APPETITE IN MAN AND THE FOOD INTAKE OF EXPERIMENTAL ANIMALS. BY DIRECT REDUCTION OF CALORIC INTAKE, THEY PROVIDE A VARIABLE AND REVERSIBLE PROCESS FOR THE TREATMENT OF OBESITY. AMONG THESE ACIDS ARE THOSE SYNTHESIZED BY THE HUMAN LIVER. NO TOXIC OR OTHERWISE UNTOWARD EFFECT ON THE CENTRAL NERVOUS SYSTEM, ON RENAL, HEPATIC OR HEMATOLOGICAL FUNCTIONS, OR ON OTHER BODY ORGANS HAS BEEN NOTED DURING THEIR USE.

United States Patent O 3,591,687 BILE ACIDS AND DERIVATIVES THEREOF ASAN ORECTIC AGENTS George A. Bray, 654 Wellesley St., Weston, Mass. 02193No Drawing. Filed Mar. 13, 1968, Ser. No. 712,583 Int. Cl. A61k 17/00US. Cl. 424-238 2 Claims ABSTRACT OF THE DISCLOSURE Certain unconjugatedbile acids as well as certain conjugates thereof, at sufiicient doselevels, suppress the appetite in man and the food intake of experimentalanimals. By direct reduction of caloric intake, they provide a variableand reversible process for the treatment of obesity. Among these acidsare those synthesized by the human liver. No toxic or otherwise untowardeffect on the central nervous system, on renal, hepatic or hematologicalfunctions, or on other body organs has been noted during their use.

BACKGROUND OF THE INVENTION ing on these phenomena hold promise becausethey may be varied either to increase or decrease the desired effects.

Hitherto, attempts at modifying the physiological mechanisms so as toreduce the appetite have generally produced insuflicient or merelytemporary effects, or have produced toxic or otherwise harmful sideeffects. Agents that produce stimulation of the central nervous systemsuch as epinephrine, dextro-amphetamine and related phenylalkylamines,reduce the appetite, but the effect has been observed to diminish over aperiod of time and there are serious side effects in the use of suchsympathomimetic amines including habituation and addiction. Theseefi'ects have not been successfully offset by the administration ofbarbiturates in conjunction with the anorectic agents.

Other attempts at weight control have involved the use of diuretics andof thyroid hormone extract or its synthetic close relatives. The latterhave also been shown to produce toxic effects resulting in nervousness,increased pulse rate and harmfully increased body metabolism.

SUMMARY OF THE INVENTION This invention consists in administeringcertain bile acids, in either unconjugated or conjugated forms andeither alone or in combinations, to reduce food intake. The mostelfective bile acids appear to be those having hydroxyl groups at twopositions with the most effective ice results obtained for the 3,7 and3,12 combinations, having reference to the following general formula.

The elfectiveness of these bile acids also appears dependent on thepresence of the C-24 carboxyl group. Effectiveness has also been foundwith the taurine and glycine conjugates of the free acids that areeffective. In these conjugates the R5 position contains the group forthe taurine and NHCH COO for the glycine.

The anorectic effectiveness of certain free bile acids characterized asabove has been demonstrated thus far in humans, and that of theconjugates in experimental animals. Effectiveness depends on the dailyoral dose levels, with thresholds for eifective lowering of the appetitein humans generally in the range above 800 milligrams. Sustained,continuous treatment at levels above the indicated threshold havedemonstrated the continuing effectiveness of this therapy in suppressingappetite in obese individuals.

Bile acids are found in nature predominately as conjugates of taurineand glycine, the free bile acids being found only in smallconcentrations in some preparations of bile. They are synthesized in theliver from cholesterol and secreted into the gastrointestinal tractwhere they are involved in the activation of pancreatic lipase and inthe absorption of fat. In this process the bile salts are secreted backinto the biliary tree by the liver and reutilized repeatedly.

The present therapy does not appreciably involve malabsorption becauselittle effect on the fecal excretion of fat is observed in its use.Little or no effect is noted in the pulse rate and body temperature andin the histology of intestinal mucosa.

DESCRIPTION OF THE PREFERRED EMBODIMENT The anorectic effect ofdeoxycholic acid (5 8-cholanic ac1d-3a,12a-diol) 1 in man was expectedlynoted in a trial The designations a and 13 represent substituentpositions nto rind out of the plane represented by the structural ormua.

designed to determine whether it would function on malabsorptionprinciples as measured by an increase in the fecal excretion of fat.This function had been previously noted in the use of cholestyramine, ananion exchange resin that binds bile acids and salts and producessteatorrhea.

In this study, each of six obese volunteer outpatients was firstinstructed in an 1800 calorie high fat diet. Gelatin capsules wereprepared, each containing 400 mg. of commercially available deoxycholicacid. The deoxycholic acid was orally administered at the rate of 1200mg. per day in three divided doses, alternating with placebos at twoweek intervals over a three-month period. Adminis tration was carriedout on a double-blind basis, that is, neither the patients nor theprescribing doctor was informed when the patients were given the bileacid-containing capsules and when they were given the placebos.

By the end of the trial period, four of the six patients hadspontaneously reported that one of the pills had suppressed appetite.Examination of the records showed that appetite suppression hadcoincided with administration of deoxycholic acid. The results aresummarized in Table 1.

TABLE 1 Patlents Age Weight Eficct on initials Sex (yrs.) (lbs) appetite21 440 Suppression.

37 275 No effect TABLE 2 Age Weight (yrs.) (lbs.)

Cholic acid {produced no effect distinguishable from the placebo. On theother hand, chenodeoxycholic acid reduced appetite in three of the fivepatients and was significantly more efiective than cholic acid (P .05)when compared by the Students t test, wherein P is an indication ofstatistical significance and measures the probability of obtaining likedata from a putative population in which the acids would be of equaleffect. Patient E. G.s food intake rose sharply after termination of thechenodeoxycholic acid treatment and she reported that her ap'petite hadincreased.

Hyodeoxycholic acid and lithocholic acid produced an effect intermediatebetween cholic and chenodeoxyc'nolic acids.

Blood test results on patient E. G. are representative of the tests onother patients and showed no changes resulting from treatment with bileacids. These results are summarized in Table 3.

TABLE 3 Caloric Bill- Choles- Bile acid intake W13 0 B UN rubin S GPT aterol K Lithoeholie *4, 446;|;106 10, 400 20 0. 1 20 9. 4 154 4. 9 4,6331129 7, 700 17 0. 1 9. 4 183 4. 8 5, 90051276 7, 250 19 0. 2 10 10. 2175 5. 0 ol 5, 67931192 8, 500 17 0. 2 20 9. 5 4. 8 Hyodeoxyeholic a,0945:1633 9, 350 is 0. 2 10 10. 0 4. 5

*Mean 5: standard error of the mean for seven day period.

WBOwhite blood count. BUNblo0d urea nitrogen.

S GPT-sernm glutamic-pyruvic transamlnase.

Ca-calcium. Kpotasslum.

Eight toxicity studies were conducted on seven volunteer hospitalizedpatients as shown by Tables 4 and 5. (One patient C. D. was involved intwo separate studies.) All eight studies included deoxycholic acid.Hyodeoxycholic acid, chenodeoxycholic acid, lithocholic acid and cholicacid were also fed to two of the patients and patient P. D. received thecombinations of bile acids listed in Table 5 for intervals of 12 dayseach. All of the bile acids were that one patient, E. G., Was studiedwhile hospitalized and administered orally in gelatin capsules.

TABLE 4 Dose Body Daily every Dura- PBI, Age weight Diet Fat Bile dose8hrs tion gm. RAT 17-0HCS Urlo Patients initials (yrs.) Sex (kg.)(calories) (percent) acid(s) (mg.) (mg) (days) percent percent FBS(mg/day) acid F.0ur 20 M 166.8 1,260 20 DC 1,200 400 12 6.9 91 25.7 10.1V 49 M 193.5 1,200 20 D 1, 200 400 12 6.3 22.4 86 13.1 9.2 24 M 131.61,288 48 13 1 4.6 33.4 75 7.0 8.7

, 4 1 0.1 (study 8 M 2,500 40 DC 1,800 600 1,600 50 DC 1,200 400 12 o.D. (study2)... 28 M 167.8 1:288 g3 g3 153g 2 6.2 7s 9.5 1,600 50 DC11200 24 1 600 10 DO 1,200 400 9 F. 06 44 F 146.9 1,600 50 DO 1.200 4009 6.1 35.8 98 25.4 7.4 1, 600 50 Do 1, 200 a) 12 1,200 400 6 1,200 400 6no 1 131.2 :83 g 4.8 26.0 82 10.1

1,200 400 6 1,200 400 6 P.D F 105.1 6.0 25.7 120 13.6 7.1

TABLE 5 Daily dose Bile acid(s) All seven patients were grossly obeseand showed the expected high values from the excretion of17-hydroxycorticosteroids. Values for uric acid were high, but only P.D. had a fasting blood sugar greater than 100. Tests of thyroid function(FBI and RAI) were within normal limits.

All patients were fed liquid formula diets with varying quantities offat added as cream.

In all eight studies deoxycholic acid produced an increase in thesecretion of sodium and chloride in the stool but did not increase theexcretion of fat, nitrogen or potassium. Feeding 1200 or 1800 mg. ofthis acid daily did not raise the level of bile acid in the blood abovenormal in C. D. (study 1) or C. D. (study 2). None of the other bileacids tested on P. D. and E. G. had any eiiect on the fecal excretion ofsodium, potassium, nitrogen or fat. Pulse rate and body temperature wereunaffected by any of the bile acids used in these toxicity studies.

Blood tests were conducted during all of the foregoing studies,including complete blood count, BUN, FBS, bilirubin, serum enzymes andelectrolytes, creatinine, alkaline phosphatase, calcium, phosphorus,total protein, albumin/ globulin ratio, iron, carotene, prothrombintime, uric acid, serum glutamic oxaloacetic transaminase, serumglutamicpyruvic transaminase, lactic dehydrogenase and xylose tolerancetests. No tests indicated abnormal results, except that the serumcarotene was depressed to the lower limits of normal in all studies, andone elevation of serum bilirubin was noted (Table 5, interval 4) whenchlolic acid was given. That elevation was not subsequently repeatedwhen the same bile acid 'was given again (interval 17).

Peroral intestinal biopsies were also obtained with the Crosby capsulebefore and during these toxicity studies at two, four and six Weeks andfive months after the beginning of treatment in five of the sevenpatients. There was no histological change in the intestinal mucosa as aresult of the giving of the bile acids.

Patients P. -D. reported significant reduction in appetite duringintervals 5, 6, 7, 8 and 9 (Table 5), which indicated not only thatdeoxychloci acid was effective when fed alone, but also that the feedingof combinations of the acids was likewise efifective to suppressappetite.

Still further tests were carried out on male albino rats weighing 200 to330 grams by intraperitoneal injections to obtain comparativemeasurements of the anorectic effects of a number of commerciallyavailable bile acids and their conjugates (sometimes referred to assalts) and of d-amphetamine sulfate. The latter is a drug widely usedfor weight control but one producing undesirable stimulation of thecentral nervous system. The compounds tested were dissolved in a vehicleconsisting of 0.1 normal aqueous solution of sodium hydroxide, dilutedto 0.15 molar aqueous solution of sodium chloride and neutralized to pH7.4. The results generally agreed with those obtained or suggested bythe human trials and included statistical comparisons using the Studentst test.

Two groups of six animals each were used, one of which was a controlgroup receiving injections of the vehicle at the same times as theexperimental group. Since there was significant variability in the foodintake from day to day within the same group of animals, all comparisonswere made with the control group. The animals were used in rotationswith none receiving more than two injections of experimental drug eachweek.

The animals 'were trained to eat their entire daily food intake within asix hour period throughout which food was made available. Tap water wasavailable ad lib. All drugs were given 30 minutes before food waspresented and the food intake for individual animals was measured eachtwo hours during the six hours when it was available.

7 d-Amphetamine was given in doses of 0.3, 3.0 and 30.0 mfg/kg. of bodyweight (i.e., 1.63, 16.3 and 163 micromoles of free base/kg). Thehighest dose reduced food intake 95 percent over the entire six hourtest period following injection, but characteristically the acuteanorectic Cholic acid had a small effect, but neither its taurine norits glycine conjugates had any effect. Lithocholic acid had a small butshort-lasting effect on appetite, but its taurine conjugate did not.

Hyodeoxycholic acid was effective in one experiment effect had subsidedby the next day. The dose of 3.0 mg./ but not in a second one. When thehydroxyl groups were kg. reduced food intake only 18 percent over theentire six oxidized to keto groups the compound produced had no hourtest period following injection. Although it produced effect onappetite.

a 91 percent reduction of food intake during the first two Introductionof a double bond at the five position, and hours, a higher than normalfood intake was noted in a 3-fi-hydroxyl group produced a compoundhaving an the second and third two-hour intervals. Also, at theinteranorexigenic effect but one not significantly different mediate andhighest dose levels some increase in motor from the control animals.

activity was observed. Two experiments on rats were carried out to testfive The doses of bile acid and conjugates were lower than bile acidswith respect to actual weight gain when the the corresponding doses ofamphetamine, i.e., 1, 10 and 1? compounds were administered over aperiod of four weeks 100 micromoles/ kg. Most of the bile acids and bilesalts or more. The bile acids were added to a semisynthetic diet causedlethargy shortly after the highest doses were inin an amount of 1.5 mg.of acid per gram of diet, this jected, but this effect promptly woreoff. The doses of 1 being the sole source of food. The acids tested weredeand 10 micromoles had little or no effect either on lethoxycholic,chenodeoxycholic, cholic, lithocholic, and hyoargy or on the inhibitionof appetite. 20 deoxycholic acid.

As with the amphetamine, the highest doses of certain During 26 days offeeding the rate of weight gain was of the bile acids and theirderivatives had an amorectic Slowed in all groups receiving bile acid,and none of the effect that wa rapid in on t, H e i t t to the bileacids had a significantly greater effect than any of the amphetamine,the effect did not subside as quickly, even rsat the intermediate doselevels. This is illustrated by the At the end of the feeding period eachanimal in all case of a single injection of chenodeoxycholic acid that oEf p Was autopsied d pieces of tissue were fixed for depressed foodintake on three consecutive days by 46, histological examination, andblOOd was drawn for labor- 48 and 38 percent, respectively. Similarprolonged effecatorY te$t$- tiveness was indicated for deoxycholic acid,glycocheno- Blood Urea nitrogen, serum glutamic oxaloacetictransdeoxycholic acid, taurochenodeoxycholic acid and tauraminase,bilifubifl and Creatinine were not affected y odeoxycholic acid. thetreatment. Carotene levels were undetectable in any The effectivenessofthe tested bile a id and th i 1 animal. Protein electrophoresis of theserum and electrorivatives is summarlzed in Table 6. phoresis forlipoproteins revealed normal patterns in all TABLE 6 G-hr. food intakeTest (percent of Name No. R1 R2 R3 R4 R5 control) Lithochollc a-OH H H HOH 71 H H H (t) 84 Hyodcoxycholic..... a-OH H H OH 90 a-OH H OHGl1ouodeoxychollc H a-OH H OH 3 55 H a-OH H 011 34 H a-OH H (t) 2 40 Ha-OH H (g) 39 H (Z-OH H OCH: 100 H (ac) H OH 103 H (ac) H OCH; 04 H (ac)H 00113 84 Ursodeoxycllolic a-OH H {3-011 H OH 1 54 Deoxycholic a-OH H Ha-OH OH 3 34 H H a-OH (t) 3 51 H H a-OH (g) 52 H H a-OH 00113 100 H a-OHa-oH 0H 3 H 01-011 02-011 (t) 98 H oz-OH a-OH (g) 88 =0 H H OH 100 H H HOH 76 H H H OH 3 72 H (ac) 00H; 1 62 H 0H 0H (24 66 H OH OH 24 83 (2;)CHECK at 24 position.

Chenodeoxycholic acid and deoxycholic acid are shown animals exceptthose receiving hydeoxycholic acid. These by the table to be highlyeffective in reducing appetite, rats showed an increase in the gammaglobulin and an and the taurine and glycine conjugates of the former areincrease in the oil 0 red staining material at the origin 'bl th hf '1'd h'l possi y even more potent an t e ree bl e acl w 1e of thellpoprotein electrophoresis strips. the taunne derivative of the latteralso effectively in- Histological examination of the intestine revealedno hibits appetite. On the other hand, when the 24 carboxyl changes.group III deoxycholic acid was esterified to produce methyl- As a resultof the foregoing trials, tests and experideoxycholate, the compoundproduced had no effect on 7 ments, it is believed that useful methodshave been disappetite. Ursodeoxycholic acid was also effective.

covered for the treatment of obesity in humans by administration of bileacids and certain derivatives and combinations thereof.

I claim:

1. A method of reducing body weight in a human through reduction in theintake of food, which consists in oral administration to a human host,in an amount between approximately 0.8 and 1.8 grams per day of acompound selected from the group consisting of deoxycholic acid,taurodeoxycholic acid, glycodeoxycholic acid, chenodeoxycholic acid,taurochenodeoxycholic acid, and glycochenodeoxycholic acid.

2. A method for reducing obesity in a human through sustained reductionin the intake of food, which consists in administering to a human hostwith the daily diet from 1.2 to 1.8 grams per day of a compound selectedfrom the group consisting of deoxycholic acid, taurodeoxycholic acid,glycodeoxycholic acid, chenodeoxycholic acid, taurochenodeoxycholicacid, and glycochenodeoxycholic 5 acid.

References Cited UNITED STATES PATENTS 3,001,986 9/1961 Burtner et a1.260239.5 10 3,153,615 10/1964 Bosshardt et a1. 424238 3,180,794 4/1965Antonides 424-238 STANLEY J. FRIEDMAN, Primary Examiner F. E. WADDELL,Assistant Examiner 2 533 UNITED STATES PATENT OFFICE CERTIFICATE OFCORRECTION Patent No. 3 59l,687 Dated July 6, 1971 Inventor(s) G orge A.Bray It is certified that error appears in the above-identified patentand that said Letters Patent are hereby corrected as shown below:

Column 2, line 60, "expectedly" should read --unexpectedly-.

Column 5 and 6, Table 4, the number "1200" under column heading "Diet(calories)" should read. --l260--;

the number "7.0" under column heading "l7-OHCS" should. read --7.l--.

Column 6, line 45, "deoxychloci" should read -deoxycholic--.

Column 7, line 22, "amorectic" should read -anorectic-;

Table 6, for hyodeoxycholic Test No. 2, column R should read --H-.

Signed and sealed this 21 st day of December I 971 SEAL Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSGHALK Attesting Officer ActingCommissioner of Batents

